Lipid analogs for treating viral infections

ABSTRACT

A method of treating viral infections, and in particular HIV-1, hepatitis B virus, and herpesviruses, is disclosed. The method comprises administering to a subject in need of such treatment an infection-combating amount of a phospholipid or phospholipid derivative.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.08/793,470, filed May 2, 1997, now U.S. Pat. No. 5,962,437 which is a371 of International Application PCT/US95/10111, filed Aug. 7, 1995,which is a continuation of application Ser. No. 08/314,901, filed Sep.29, 1994, now abandoned, which is a Continuation-In-Part of applicationSer. No. 08/297,416, filed Aug. 29, 1994, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to the treatment of viral infections,and more specifically to the treatment of viral infections withphospholipids and phospholipid derivatives.

BACKGROUND OF THE INVENTION

A current treatment for combating human immunodeficiency virus type 1(HIV-1) infections is the administration of the nucleoside analog3′-azido-3′-deoxythymidine (AZT) to an afflicted subject. See, e.g.,U.S. Pat. No. 4,724,232 to Rideout et al. HIV-1 infection treatmentmethods have also included the administration of ether lipid compoundsin an amount effective to inhibit replication of the virus in infectedcells, see, e.g., Kucera et al., AIDS Research and Human Retroviruses6:491 (1990), and ether lipids conjugated with AZT and other antiviralnucleoside analogs. See PCT Application No. US91/04289 (published 26Dec. 1991). These compounds appear to act at the plasma membrane toblock the endocytic process of HIV-1 into CD4⁺ cells and the process ofvirus assembly, cell fusion and pathogenesis. They also can inhibit theactivity of protein kinase C. Given the seriousness of HIV-1 infectionworldwide, there is an ongoing need for new methods of combating HIV-1infections.

Another virus of serious concern, hepatitis B virus (HBV), is one of afamily of hepadnaviruses that cause acute and chronic liver disease,including liver cancer. HBV, which is found in the body fluids ofinfected persons, makes three antigenic proteins during multiplicationin liver cells: hepatitis B surface antigen (HBsAg), hepatitis B eantigen (HBeAg) and hepatitis B core antigen (HBcAg). These three virusantigenic proteins are important as markers for determining virusinfection, as antibodies against the virus infection are made inresponse to these virus proteins in the blood. An HBV vaccine isavailable to prevent infection, and hyperimmune gamma globulin isavailable for temporary prophylaxis against developing HBV infection inpersons at risk. Clearly specific antiviral agents are needed fortreatment and control of HBV infections in humans.

Based on the foregoing, it is an object of the present invention toprovide a new treatment method for combating the effects of HIV-1.

It is another object of the present invention to provide compounds andpharmaceutical compositions for carrying out HIV-1 treatment methods.

It is also an object of the present invention to provide a new treatmentmethod for combating the effects of HBV.

It is a second object of the present invention to provide compounds andpharmaceutical compositions for carrying out HBV treatment methods.

SUMMARY OF THE INVENTION

These and other objects are satisfied by the present invention, whichprovides methods of combating viral infections. As a first aspect, thepresent invention provides a method of combating a viral infection in asubject in need of such treatment comprising administering to thesubject an effective infection-combating amount of a compound of FormulaI or a pharmaceutical salt thereof.

In the compounds of Formula I, R₁ is a branched or unbranched, saturatedor unsaturated C₆ to C₁₈ alkyl group optionally substituted from 1 to 5times with —OH, —COOH, oxo, amine, or substituted or unsubstitutedaromatic; X is selected from the group consisting of NHCO, CH₃NCO, CONH,CONCH₃, S, SO, SO₂, O, NH, and NCH₃; R₂ is a branched or unbranched,saturated or unsaturated C₆ to C₁₄ alkyl group optionally substitutedfrom 1 to 5 times with —OH, —COOH, oxo, amine, or substituted orunsubstituted aromatic; Y is selected from the group consisting of NHCO,CH₃NCO, CONH, CONCH₃, S, SO, SO₂, O, NH, and NCH₃; R₆ is a branched orunbranched C₂ to C₆ alkyl group; and R₃, R₄, and R₅ are independentlymethyl or ethyl, or R₃ and R₄ together form an aliphatic or heterocyclicring having five or six members and R₅ is methyl or ethyl. Preferredcompounds include 1-dodecanamido-2-decyloxypropyl-3-phosphocholine,1-dodecanamido-2-octyloxypropyl-3-phosphocholine, and1-dodecanamido-2-dodecyloxypropyl-3-phosphocholine. The method isparticularly preferred as a treatment to combat viral infections causedby HIV-1, HBV, and herpes simplex virus. The present invention alsoincludes pharmaceutical compositions comprising a compound of Formula Iand a suitable pharmaceutical carrier.

As a second aspect, the present invention includes a method of combatingviral infections in a subject in need of such treatment which comprisesthe administration to such a subject a compound of Formula II or apharmaceutical salt thereof in an effective infection-combating amount.

In Formula II, the ring structure is optionally substituted from 1 to 3times with C₁ to C₃ alkyl; R₁ is an unbranched or branched, saturated orunsaturated C₆ to C₂₀ alkyl group; R₂, R₃, and R₄ are independentlymethyl or ethyl, or R₂ and R₃ together form an aliphatic or heterocyclicring having five or six members and R₃ is methyl or ethyl; X is selectedfrom the group consisting of NHCO, CH₃NCO, CONH, CONCH₃, S, SO, SO₂, O,NH, and NCH₃; R₅ is a branched or unbranched C₂ to C₆ alkyl group; m is1 to 3; and n is 0 to 2. Preferred compounds of Formula II are3-hexadecanamido-cyclohexylphosphocholine and3-hexadecylthio-cyclohexylphosphocholine. Adminstration of the compoundsof Formula II is particularly useful in treating viral infections causedby HIV-1, HBV, and herpesviruses. The present invention also includespharmaceutical compositions comprising a compound of Formula II and asuitable pharmaceutical carrier.

A third aspect of the present invention is a method of treating viralinfections comprising administering to a subject in need of suchtreatment an effective infection-inhibiting amount of a compound ofFormula III.

In compounds of Formula III, R₁ is a branched or unbranched, saturatedor unsaturated C₆ to C₁₈ alkyl group optionally substituted from 1 to 5times with —OH, —COOH, oxo, amine, or substituted or unsubstitutedaromatic; X is selected from the group consisting of NHCO, CH₃NCO, CONH,CONCH₃, S, SO, SO₂, O, NH, and NCH₃; R₂ is a branched or unbranched,saturated or unsaturated C₆ to C₁₄ alkyl group optionally substitutedfrom 1 to 5 times with —OH, —COOH, oxo, amine, or substituted orunsubstituted aromatic; Y is selected from the group consisting of NHCO,CH₃NCO, CONH, CONCH₃, S, SO, SO₂, O, NH, and NCH₃; and Z is a moiety ofthe Formula V,

wherein:

V is H or N₃;

W is H or F; or

V and W together are a covalent bond; and

B is a purinyl moiety of Formula VI

optionally substituted at position 2 with ═O —OH, —SH, —NH₂, or halogen,at position 4 with NH₂ or ═O, at position 6 with Cl, —NH₂, —OH, or C₁–C₃alkyl, and at position 8 with Br or I; or

B is a pyrimidinyl moiety of Formula VII

substitued at position 4 with ═O or NH₂ and optionally substituted atposition 5 with halogen or C₁–C₃ saturated or unsaturated alkyloptionally substituted 1 to 3 times with halogen.

Pharmaceutical compositions comprising these compounds and apharmaceutical carrier are also encompassed by the present invention.

A fourth aspect of the invention is a method of inhibiting viralinfections comprising administering to a subject in need of suchtreatment an effective infection-inhibiting amount of a compound ofFormula IV.

In the compounds of Formula IV, the ring structure is optionallysubstituted from 1 to 3 times with C₁ to C₃ alkyl; R₁ is an unbranchedor branched, saturated or unsaturated C₆ to C₂₀ alkyl group; X isselected from the group consisting of NHCO, CH₃NCO, CONH, CONCH₃, S, SO,SO₂, O, NH, and NCH₃; m is 1 to 3; n is 0 to 2; and Z is a moiety of theFormula V,

wherein:

V is H or N₃;

W is H or F; or

V and W together are a covalent bond; and

B is a purinyl moiety of Formula VI

optionally substituted at position 2 with ═O —OH, —SH, —NH₂, or halogen,at position 4 with NH₂ or ═O, at position 6 with Cl, —NH₂, —OH, or C₁–C₃alkyl, and at position 8 with Br or I; or

B is a pyrimidinyl moiety of Formula VII

substitued at position 4 with ═O or NH₂ and optionally substituted atposition 5 with halogen or C₁–C₃ saturated or unsaturated alkyloptionally substituted 1 to 3 times with halogen.

The present invention also includes pharmaceutical compositionscomprising a compound of Formula IV and a suitable pharmaceuticalcarrier.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “alkyl” is intended to refer to an unbranchedor branched alkyl group comprising carbon atoms, such as methyl, ethyl,propyl, isopropyl, n-butyl, tert-butyl, hexyl, and octyl. The term“pharmaceutical salt” refers to a salt that retains the desiredbiological activity of the parent compound and does not impart undesiredtoxicological effects thereto. Examples of such salts are (a) saltsformed with cations such as sodium, potassium, NH₄ ⁺, magnesium, calciumpolyamines, such as spermine, and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

A first aspect of the present invention is a method of combating viralinfection comprising administering a compound of Formula I, wherein R₁,R₂, R₃, R₄, R₅, R₆, X, and Y are defined as stated above, or apharmaceutical salt thereof. The amphipathic compounds of Formula I,which are generally analogs of phosphatidylcholine, include a glycerolbackbone (represented by the chain of three carbon atoms to which otherfunctional groups are bonded), lipophilic moieties (represented by R₁and R₂) bonded to positions 1 and 2 of the glycerol backbone throughfunctional groups (represented by X and Y) that are generally resistantto phospholipase degradation, and polar phosphate and quaternary aminegroups (linked to one another through a short alkyl group) bonded toposition 3 of the glycerol backbone. Each of these components of thecompounds of Formula I is described separately below.

In Formula I, as described above, R₁ is a lipophilic moiety; thelipophilicity of R₁ allows the compounds of Formula I to bind with thecell membrane of a cell infected with a retrovirus to provide an anchorthereto. R₁ can be an unbranched or branched, saturated or unsaturatedC₆ to C₁₈ alkyl group. Preferably, R₁ is an unbranched saturated orunsaturated C₈ to C₁₂ alkyl group, and more preferably, R₁ is anunbranched saturated C₁₀ or C₁₂ alkyl group.

In compounds of Formula I, X is a functional group that links thelipophilic moiety R₁ and the glycerol backbone of the compound. X isselected from the group consisting of NHCO, CH₃NCO, CONH, CONCH₃, S, SO,SO₂, O, NH, and NCH₃; these functional groups are resistant to thehydrolytic activity of cellular lipases, in particular phospholipase A,which is specific for ester linkages at position 1 (as are present inphosphatidyl choline). Preferably, X is S or NHCO, with NHCO being mostpreferred.

In Formula I, R₂ is a lipophilic moiety which, as is true for R₁,enables the compounds of Formula I to bind with the cell membrane of aninfected cell. R₂ can be an unbranched or branched, saturated orunsaturated C₆ to C₁₄ alkyl group. Preferably, R₂ is an unbranchedsaturated or unsaturated C₈ to C₁₂ alkyl group, and more preferably, R₂is an unbranched saturated C₈ or C₁₀ alkyl group. It is also preferredthat R₁ and R₂ together contain between 18 and 22 carbon atoms.

R₂ is bonded to position 2 of the glycerol backbone through a functionalgroup Y, which is selected from the group consisting of NHCO, CH₃NCO,CONH, CONCH₃, S, SO, SO₂, O, NH, and NCH₃. Like X, Y should be a moietythat is resistant to the hydrolytic activity of cellular lipases, and inparticular phospholipase B, as this enzyme is specific for esterlinkages at position 2. Preferably, X is S or O, with O being morepreferred.

The polar hydrophilic end of the amphipathic compounds of Formula I,which can play a role in membrane interaction, comprises an amphotericphosphoalkyl quaternary amine group in which the phosphate moietycarries the negative charge and the quaternary amine moiety carries thepositive charge. In this group, R₆, which is a branched or unbranched,saturated or unsatured C₂ to C₆ alkyl group, is preferably saturated C₂.R₃, R₄, and R₅ are independently selected from the group consisting ofmethyl and ethyl, with methyl being preferred, and with R₃, R₄, and R₅each being methyl being more preferred, or R₃ and R₄ together form analiphatic or heterocyclic ring having five or six members and R₅ ismethyl or ethyl.

Exemplary compounds of Formula I include1-dodecanamido-2-decyloxypropyl-3-phosphocholine (CP-128),1-dodecanamido-2-octyloxypropyl-3-phosphocholine (CP-130),1-dodecanamido-2-dodecyloxypropyl-3-phosphocholine (CP-131), and1-dodecyloxy-2-decyloxypropyl-3-phosphocholine (CP-129). These compoundsof Formula I can be synthesized according to the procedures set forth inExamples 1 and 2 below. Other compounds of Formula I can be synthesizedusing the same method with the appropriate reagents substituted forthose listed.

Another aspect of the invention is a method of combating viral infectionby administering compounds of Formula II, wherein R₁, R₂, R₃, R₄, R₅, X,m, and n are defined as stated above, or a pharmaceutical salt thereof.Compounds of Formula II are amphipathic-moieties having a lipophilicmoiety (represented by R₁)-linked to a five- or six-membered ringstructure (which is optionally sustituted 1 to 3 times with C₁ to C₃alkyl) and a hydrophilic moiety that includes phosphate and quaternaryamine groups linked by a short alkyl group that is bonded to the ringstructure through the phosphate group. The hydrophilic group is linkedto the ring at position 1, and the lipophilic group is linked to thering at positions 2, 3, or 4. Like the compounds of Formula I, thecompounds of Formula II are analogs of phosphatidyl choline. However,the ring structure provides a more conformationally restricted frameworkfor the compound than compounds lacking a ring structure; thisrestricted framework can provide the compound with more favorableinteraction with the cellular membrane and thereby increase itsefficacy.

In the compounds of Formula II, R₁ can be an unbranched or branched,saturated or unsaturated C₆ to C₂₀ alkyl group. As with the compounds ofFormulas II, R₁ is a lipophilic moiety which binds with the cellmembrane of infected cells to provide an anchor thereto. Preferably, R₁is unbranched saturated or unsaturated C₁₀ to C₁₈ alkyl. Morepreferably, R₁ is unbranched saturated or unsaturated C₁₆ to C₁₈ alkyl.

In compounds of Formula II, X is a functional group that links thelipophilic moiety R₁ to position 1 of the ring structure. X should be afunctional group, such as NHCO, CH₃NCO, CONH, CONCH₃, NH, NCH₃, S, SO,SO₂, or O, that is able to withstand the hydrolytic activity of cellularlipases. Preferably, Y is S or NHCO.

As stated above, the polar hydrophilic end of the amphipathic compoundsof Formula II comprises a phosphate group bonded to the ring structure,a short alkyl group R₅ linked at one end thereto, and a quaternary aminegroup linked to the opposite end of the short alkyl group. R₅ is asaturated or unsaturated, branched or unbranched C₂ to C₆ alkyl group,and is more preferably C₂. R₂, R₃, and R₄ are independently selectedfrom the group consisting of methyl and ethyl, with methyl beingpreferred, or R₂ and R₃ together form an aliphatic or heterocyclic five-or six-membered ring structure and R₄ is methyl or ethyl. It is morepreferred that R₂, R₃, and R₄ are each methyl.

In the compounds of Formula II, m can be 1, 2, or 3, and n can be 0, 1,or 2. Preferably the ring structure is a five- or six-membered ring;thus, preferably m is 2 or 3 when n is 0, m is 1 or 2 when n is 1, and mis 1 when n is 2. As noted above, the ring structure providesconformational rigidity to the compound.

Exemplary compounds of Formula II include3-hexadecylthio-cyclohexylphosphocholine (INK-1),3-hexadecanamido-cyclohexylphosphocholine.3-hexadecanamido-cyclopentylphosphocholine, and3-hexadecylthio-cyclopentylphosphocholine. These compounds of Formula IIcan be synthesized by following the teachings of Example 3 below incombination with procedures known to those skilled in the art.

An additional aspect of the present invention is a method of combatingviral infection with compounds of Formulas III and IV. These compoundssubstitute a moiety Z for the alkyl-quaternary amine of the compounds ofFormulas I and II, wherein Z is as defined above. Z is a moiety that hasdemonstrated anti-viral activity by itself; thus conjugation of Z to theremainder of the compounds of Formulas III and IV provides a compoundthat potentially includes multiple active sites for viral inhibition.

In the compounds of Formula III, R₁, R₂, X and Y are defined above. R₁is a lipophilic moiety; the lipophilicity of R₁ allows the compounds ofFormula I to bind with the cell membrane of a cell infected with aretrovirus to provide an anchor thereto. R₁ can be an unbranched orbranched, saturated or unsaturated C₆ to C₁₈ alkyl group. Preferably, R₁is an unbranched saturated or unsaturated C₈ to C₁₂ alkyl group, andmore preferably, R₁ is an unbranched saturated C₁₀ or C₁₂ alkyl group.

In compounds of Formula III, X is a functional group that links thelipophilic moiety R₁ and the glycerol backbone of the compound. X isselected from the group consisting of NHCO, CH₃NCO, CONH, CONCH₃, S, SO,SO₂, O, NH, and NCH₃; these functional groups are resistant to thehydrolytic activity of cellular lipases, in particular phospholipase A,which is specific for ester linkages at position 1 (as are present inphosphatidyl choline). Preferably, X is S or NHCO, with NHCO being mostpreferred.

In Formula III, R₂ is a lipophilic moiety which, as is true for R₁,enables the compounds of Formula III to bind with the cell membrane ofan infected cell. R₂ can be an unbranched or branched, saturated orunsaturated C₆ to C₁₄ alkyl group. Preferably, R₂ is an unbranchedsaturated or unsaturated C₈ to C₁₂ alkyl group, and more preferably, R₂is an unbranched saturated C₈ or C₁₀ alkyl group. It is also preferredthat R₁ and R₂ together contain between 18 and 22 carbon atoms.

R₂ is bonded to position 2 of the glycerol backbone through a functionalgroup Y, which is selected from the group consisting of NHCO, CH₃NCO,CONH, CONCH₃, S, SO, SO₂, O, NH, and NCH₃. Like X, Y should be a moietythat is resistant to the hydrolytic activity of cellular lipases, and inparticular phospholipase B, as this enzyme is specific for esterlinkages at position 2. Preferably, X is S or O, with O being morepreferred.

In the compounds of Formula III, Z is a moiety of Formula V. Moieties ofFormula V are intended to be anti-viral agents, and thus potentiallyprovide an additional active site for anti-viral activity that may actthrough a different mechanism. In the moieties of Formula V, V is H, orN₃, or V and W together from a covalent bond with H and N₃ beingpreferred. W is H or F, with H being preferred.

In the compounds of Formula III, B is a purinyl moiety of Formula VI ora pyrimidinyl moiety of Formula VII, each of which are substituted asdescribed above. As used herein, a purinyl moiety comprises six- andfive-membered aromatic rings having the molecular structure illustratedin Formula VI. Those skilled in this art will appreciate that the doublebonds illustrated in Formula VI are present to represent that thepurinyl moieties have aromatic character, and that these double bondsmay shift their positions in certain compounds due to the presence ofcertain substituents to retain the aromatic character of the moiety; inparticular, those moieties having ═O or NH₂ substituents at positions 2and 4, such as adenine, guanine, xanthine, and hypoxanthine, aregenerally illustrated as having double bonds shifted from the positionsshown in Formula VI. Similarly, as used herein a pyrimidinyl moietycomprises a six-membered aromatic ring having the molecular structureillustrated in Formula VII. Those skilled in this art will appreciatethat the double bonds illustrated in Formula VII are included therein torepresent that the moieties of Formula VII have aromatic character, andthat these double bonds may shift for certain substituents, inparticular for ═O and NH₂ at positions 2 and 4, in order for the moietyto retain its aromatic character. Preferably, B is selected from thegroup consisting of adenine, thymine, cytosine, guanine, hypoxanthine,uracil, 5-fluorouracil, 2-fluoro-adenine, 2-chloro-adenine,2-bromo-adenine, and 2-amino-adenine.

Preferably, Z is 3′-azido-3′-deoxythymidine, dideoxyinosine,dideoxycytidine, or 2′, 3′-didehydro-3′-deoxythymidine. An exemplarypreferred compound of Formula III is3′-azido-3′-deoxy-5′-(3-dodecanamido-2-decyloxypropyl)-phosphothymidine.

A further aspect of the present invention is a method of inhibitingviral infections comprising administering to a subject an effectiveinfection-inhibiting amount of a compound of Formula IV, wherein R₁, R₂,X, m, n, and Z are as defined above. In the compounds of Formula IV, R₁can be an unbranched or branched, saturated or unsaturated C₆ to C₂₀alkyl group. As with the compounds of Formula II, R₁ is a lipophilicmoiety which binds with the cell membrane of infected cells to providean anchor thereto. Preferably, R₁ is unbranched saturated or unsaturatedC₁₀ to C₁₈ alkyl. More preferably, R₁ is unbranched saturated orunsaturated C₁₆ to C₁₈ alkyl.

In compounds of Formula IV, X is a functional group that links thelipophilic moiety R₁ to position 1 of the ring structure. X should be afunctional group, such as NHCO, CH₃NCO, CONH, CONCH₃, NH, NCH₃, S, SO,SO₂, or O, that is able to withstand the hydrolytic activity of cellularlipases. Preferably, X is S or NHCO.

As stated above, the polar hydrophilic end of the amphipathic compoundsof Formula IV comprises a phosphate group bonded to the ring structureand a moiety Z as defined in Formula V. In the moieties of Formula V, Vis H, or N₃, or V and W together form a covalent bond, with H and N₃being preferred. W is H or F, with H being preferred.

In the compounds of Formula IV, B is a purinyl moiety of Formula VI or apyrimidinyl moiety of Formula VII, each of which are substituted asdescribed above. As used herein, a purinyl moiety comprises six- andfive-membered aromatic rings having the molecular structure illustratedin Formula VI. Those skilled in this art will appreciate that the doublebonds illustrated in Formula VI are present to represent that thepurinyl moieties have aromatic character, and that these double bondsmay shift their positions in certain compounds due to the presence ofcertain substituents to retain the aromatic character of the moiety; inparticular, those moieties having ═O or NH₂ substituents at positions 2and 4, such as adenine, guanine, xanthine, and hypoxanthine, aregenerally illustrated as having double bonds shifted from the positionsshown in Formula VI. Similarly, as used herein a pyrimidinyl moietycomprises a six-membered aromatic ring having the molecular structureillustrated in Formula VII. Those skilled in this art will appreciatethat the double bonds illustrated in Formula VII are included therein torepresent that the moieties of Formula VII have aromatic character, andthat these double bonds may shift for certain substituents, inparticular for ═O and NH₂ at positions 2 and 4, in order for the moietyto retain its aromatic character. Preferably, B is selected from thegroup consisting of adenine, thymine, cytosine, guanine, hypoxanthine,uracil, 5-fluorouracil, 2-fluoro-adenine, 2-chloro-adenine,2-bromo-adenine, and 2-amino-adenine.

Preferably, Z is selected from the group consisting of3′-azido-3′-deoxythymidine, dideoxyinosine, dideoxycytidine, and 2′,3′-didehydro-3′-deoxythymidine.

In the compounds of Formula IV, m can be 1, 2, or 3, and n can be 0, 1,or 2. Preferably, the ring structure is a five- or six-membered ring;thus m is 2 or 3 when n is 0, m is 1 or 2 when n is 1, and m is 1 when nis 2. The ring structure provides conformational rigidity to thecompound.

An exemplary compound of Formula IV is3′-azido-3′-deoxy-5′-(3-hexadecylthiocyclohexyl)-phosphothymidine.

Experimentation has demonstrated the efficacy of the compounds ofFormulas I, II, III and IV in combating viral infection. For example,compounds CP-128, CP-129, CP-130, CP-131, and INK-1 in nanomolarconcentration substantially inhibit the HIV-1 activity in CEM-SS cells.Further, these compounds did so at noncytotoxic levels, thus indicatingtheir promise as therapeutic agents for treatment of viral infections.The compounds of Formulas I, II, III and IV are believed to attach tothe cell membrane and thus are particularly effective against infectionscaused by membrane-containing or envelope-containing viruses, as theseviruses typically require access to the cell membrane to multiply andassemble through the manufacture of new viral particles. For example,the compounds of Formulas I, II, III and IV can inhibit the transportand/or incorporation of HIV-1 major glycoprotein gp12O in the cellmembrane of an infected cell prior to viral assembly. Such inhibitioncan block the transmission of infectious HIV-1 into neighboring cells.In addition, compounds of Formulas I, II, III and IV can inhibit theproduction of the HBV core and “e” antigens, each of which contribute tothe assembly of new virus particles and the spread of HBV infection.Other infections for which the compounds of Formulas I, II, III and IVshould be efficious include those caused by other membrane-containing orenvelope-containing herpesviruses, influenza, respiratory syncytialvirus, mumps, measles, and parainfluenza viruses.

Experimentation has also shown that the compounds of Formulae I, II,III, and IV have potent anti-tumor activity. In particular, some ofthese compounds have IC₅₀ values of approximately 1.2 μM against theKB-cell line.

In the manufacture of a medicament according to the invention,hereinafter referred to as a “formulation,” the compounds of Formulas I,II, III and IV are typically admixed with, among other things, anacceptable carrier. The carrier must, of course, be acceptable in thesense of being compatible with any other ingredients in the formulationand must not be deleterious to the patient. The carrier may be a solidor a liquid, or both, and is preferably formulated with the compound asa unit-dose formulation, for example, a tablet, which may contain from0.5 percent to 95 percent by weight of the active compound. One or moreactive compounds may be incorporated in the formulations of theinvention, which may be prepared by any of the well known techniques ofpharmacy consisting essentially of admixing the components.

The formulations of the invention include those suitable for oral,rectal, topical, intrathecal, buccal (e.g., sub-lingual), parenteral(e.g., subcutaneous, intramuscular, intradermal, or intravenous) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or nonaqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above).

Suitable solid diluents or carriers for the solid oral pharmaceuticaldosage unit forms are selected from the group consisting of lipids,carbohydrates, proteins and mineral solids, for example, starch,sucrose, lactose, kaolin, dicalcium phosphate, gelatin, acacia, cornsyrup, corn starch, talc and the like.

Capsules, both hard and soft, are filled with compositions of theseactive ingredients in combination with suitable diluents and excipients,for example, edible oils, talc, calcium carbonate and the like, and alsocalcium stearate.

In general, the formulations of the invention are prepared by uniformlyand intimately admixing the active compound with a liquid or finelydivided solid carrier, or both, and then, if necessary, shaping theresulting mixture. For example, a tablet may be prepared by compressingor molding a powder or granules containing the active compound,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the compound in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets may be made by molding, in a suitable machine,the powdered compound moistened with an inert liquid binder.

Liquid preparations for oral administration are prepared in water oraqueous vehicles which advantageously contain suspending agents, forexample, methylcellulose, acacia, polyvinylpyrrolidone, polyvinylalcohol and the like.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavored base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin, glycerin, sucrose, or acacia.

Formulations of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of theactive compound, which preparations are preferably isotonic with theblood of the intended recipient. These preparations are preferablyadministered intravenously, although administration may also be effectedby means of subcutaneous, intramuscular, intrathecal, or intradermalinjection. The formulation should be sufficiently fluid that for easyparental administration. Such preparations may conveniently be preparedby admixing the compound with water or a glycine buffer and renderingthe resulting solution sterile and isotonic with the blood. Suchpreparations should be stable under the conditions of manufacture andstorage, and ordinarily contain in addition to the basic solvent orsuspending liquid, preservatives in the nature of bacteriostatic andfungistatic agents, for example, parabens, chlorobutanol, benzylalcohol, phenol, thimerosal, and the like. In many cases, it ispreferable to include osmotically active agents, for example, sugars orsodium chloride in isotonic concentrations. Injectable formulationsaccording to the invention generally contain from 0.1 to 5 percent w/vof active compound and are administered at a rate of 0.1 ml/min/kg.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanolin,polyethylene glycols, alcohols, and combinations of two or more thereof.The active compound is generally present at a concentration of from 0.1to 15 percent w/w, for example, from 0.5 to 2 percent w/w.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound as an optionally buffered aqueoussolution of, for example, 0.1 to 0.2M concentration with respect to thesaid active compound.

Formulations suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Pharmaceutical Research 3(6), 318, (1986)) and typically take the form of an optionally bufferedaqueous solution of the active compound. Suitable formulations comprisecitrate or bis\tris buffer (pH 6) or ethanol/water and contain from 0.1to 0.2 M active ingredient.

The compounds of Formulas I, II, III and IV are administered in anamount sufficient to combat viral infection. The dose can vary dependingon the compound selected for administration, the subject, the route ofadministration, and other factors. Preferably, the compound isadministered in an amount of at least 0.1 ng/kg, 1 ng/kg, 0.001 μg/kg ormore, and is adminstered in an amount no greater than 0.1 g/kg, 0.01g/kg, 1 mg/kg, or less.

The invention is illustrated in greater detail in the followingnonlimiting examples. In the Examples, “g” means grams, “mg” meansmilligrams, “μg” means micrograms, “μM” means micromolar, “mL” meansmilliliters, “°C” means degrees Celsius, “THF” means tetrahydrofuran,“DMF” means dimethylformamide, “mol” means moles, “mmol” meansmillimoles, and “psi” means pounds per square inch.

EXAMPLE 1 Preparation of Amidoalkyl Derivatives

The procedure set forth below was used to prepare the followingcompounds:

(a) 1-dodecanamido-2-decyloxypropyl-3-phosphocholine (CP-128)

(b) 1-dodecanamido-2-octyloxypropyl-3-phosphocholine (CP-130)

(c) 1-dodecanamido-2-dodecyloxypropyl-3-phosphocholine (CP-131)

3-Amino-1,2-propanediol was reacted with lauroyl chloride at roomtemperature in pyridine and dimethyl formamide. The resultingdodecanamido propanediol was recrystallized from chloroform, thenreacted with triphenylmethyl chloride. The tritylated product wasrecrystallized from hexanes. The C-2 hydroxyl was alkylated by reactionwith sodium hydride and the appropriate alkyl bromide in tetrahydrofuranfor formation of the ether linkage at C-2 (1-bromodecane for CP-128;1-bromooctane for CP-130; 1-bromododecane for CP-131). Columnchromatography on silica gel with a discontinuous gradient ofhexanes:ethyl acetate (95:5 to 80:20) produced the desired1-dodecanamido-2-alkoxy-3-trityloxypropane. Detritylation withp-toluensulfonic acid in 5:1 methylene chloride:methanol gave producthaving a free primary hydroxyl after column chromatography(hexanes:ethyl acetate 95:5 to 0:100). Reaction with 2-bromoethylphosphodichloridate in diethyl ether and pyridine produced the phosphateester, which was purified on silica gel with chloroform:methanol (100:0to 2:1). Displacement of the bromide with aqueous trimethylamine inchloroform:isopropanol:dimethyl formamide (3:5:5) gave the finalphosphocholine product after column chromatography withchloroform:methanol:ammonium hydroxide (70:35:1 to 70:35:7).

EXAMPLE 2 Preparation of 1-dodecyloxy-2-decyloxypropyl-3-phosphocholine(CP-129)

Isopropylidene glycerol was alkylated using potassium hydroxide and1-bromododecane in toluene. The resulting ketal was hydrolyzed withhydrochloric acid in methanol, and the diol formed thereby wasrecrystallized from methanol. The remaining reaction steps (tritylation,alkylation, detritylation, phosphorylation, amination) followed theprocedures described above in Example 1 for the alkylamido derivatives.

EXAMPLE 3 Preparation of cis- andtrans-3-hexadecylthio-cyclohexylphosphocholine (INK-1)

2-Cyclohexenone (0.14 mol, 13.4 mL) was dissolved in 10 mL of 10 percentsodium hydroxide and 50 mL of THF. An equimolar amount of hexadecylmercaptan (0.14 mol, 42.9 mL) was added to the unsaturated ketone andthe mixture refluxed to produce 3-hexadecylthiocyclohexanone (70 percentyield). This product (5.23 mmol, 1.851 g) was dissolved in methanol andreduced with sodium borohydride (5.23 mmol, 0.199 g) to give a racemicmixture of 3-hexadecylthiocyclohexanol (yield 62 percent; cis:transratio 4:1). The phosphorylating agent was prepared by refluxingphosphorus oxychloride (0.65 mol, 60.8 mL) and 2-bromoethanol (0.38 mol,27.0 mL) in 25 mL of trichloroethylene to produce 2-bromoethyldichlorophosphate (yield 53 percent). The 3-hexadecylthiocyclohexanol(0.56 mmol, 0.200 g) was dissolved in diethyl ether:THF (2:1) andrefluxed with the 2-bromoethyl dichlorophosphate (222 mmol, 0.3 mL) toproduce 3-hexadecylthiocyclohexyl phosphoethyl bromide (yield 54percent). The latter (0.276 mmol, 0.150 g) was dissolved in isopropylalcohol chloroform:DMF (5:3:5) and heated at 65° C. with trimethylamine(0.042 mol, 2 mL) to produce the desired product,3-hexadecylthiocyclohexyl-phosphocholine (yield 38 percent).

This procedure can also be used to prepare 3-alkylthio-cyclopentylderivatives by substituting 2-cyclopentenone.

EXAMPLE 4 Preparation of cis- andtrans-3-hexadecanamido-cyclohexylphosphocholine

2-Cyclohexenone is reacted with benzylamine to give3-benzylaminocyclohexanone. Hydrogenolysis of the benzylamino group thengives 3-aminocyclohexanone. Reaction with hexadecanoyl chloride affords3-hexadecanamidocyclohexanone, which is then reduced with sodiumborohydride to produce a cis/trans mixture of3-hexadecanamidocyclohexanol. Separation by column chromatography thengives the pure isomers. Reaction with bromoethylphosphodichloridate,then with trimethylamine will produce3-hexadecanamido-cyclohexylphosphocholine.

Synthesis of the 2- and 4-alkylamido derivatives can be carried outfollowing essentially similar procedures with the substitution ofappropriate starting materials.

EXAMPLE 5 Preparation of3′-azido-3′deoxy-5′-(dodecanamido-2-decyloxypropyl)-phosphothymidine

3-Dodecanamido-2-decyloxy-propanol was synthesized via the schemedescribed in Morris-Natschke et al., J. Med. Chem., 29:2114 (1986). Thisalcohol was phosphorylated with diphenylchlorophosphate in pyridine togive the corresponding phosphate ester. The phenyl groups were thenremoved via hydrolysis with PtO₂. The phosphatidic acid derivatives werethen conjugated to the 5′-hydroxyl of AZT (DCC condensation).

EXAMPLE 6 Preparation of3′-azido-3′deoxy-5′-(dodecyloxy-2-decyloxypropyl)-phosphothymidine

A. 3-Dodecyloxy-1,2-propanediol¹ Section “A” titled“3-Dodecyloxy-1,2-propanediol” appears in the originally filedspecification at page 21, line 13 as underlined text. Accordingly, theunderlining presented here in this amendment, for this part of thespecification, should not be construed as a change to page 21, line 13of the specification.

Isopropylidineglycerol (solketal, 26.4 g, 0.20 mol) in 60 mL of toluenewas added dropwise to a solution of powdered KOH (22.4 g, 0.04 mol) in150 mL toluene. The resulting mixture was refluxed for 4 hours.1-Bromodecane (50 g, 0.20 mol) in 40 mL of toluene was then addeddropwise, and the solution was refluxed for 10 hours. After cooling, thereaction mixture was diluted with 200 mL of ice-water and extracted withdiethyl ether (3×100 mL). The ether layers were dried over magnesiumsulfate, and the solvent was removed in vacuo. The residue was dissolvedin 60 mL of diethyl ether and 260 mL of MeOH. Concentrated HCl (60 mL)was added, and the solution was refluxed for 16 hours. After cooling,ice water (150 mL) was added, and the layers were separated. The aqueouslayer was extracted with diethyl ether (2×75 mL). The combined organicfractions were then dried over sodium sulfate, filtered, andconcentrated in vacuo. The solid residue was recrystallized from MeOH togive 37 g (0.14 mol), 71% of a white solid.

B. 3-Dodecyloxy-1-triphenylmethoxy-2-propanol

The diol synthesized in Section A was tritylated with trityl chloride(59 g, 0.21 mol) in pyridine (200 mL) at 70° C. for 5 hours and then atroom temperature overnight. The pyridine was removed under vacuum, andthe solid residue was partitioned between water and CHCl₃. The CHCl₃layer was washed with 5 percent HCl and water, then dried over magnesiumsulfate. After removal of solvent, the product was recrystallized fromhexanes:ethyl acetate (10:1) to give 19 g of pure product.

C. 3-Dodecyloxy-2-decyloxy-1-triphenylmethoxypropane

The trityl ether of Section B (13.5 g, 0.027 mol) was added dropwise toan ice-cooled suspension of sodium hydride (80%, 1.6 g, 0.054 mol) in150 mL of tetrahydrofuran under nitrogen. After stirring for 2 hours atroom temperature, heat was applied (55° C.). 1-Bromodecane (6 g, 0.027mol) was added dropwise; heating was continued for 6 hours. Aftercooling for 3 hours, water was added slowly. Diethyl ether (2×100 mL)was added, and the solution washed with 15 percent sodium thiosulfite,water, and brine. After drying over sodium sulfate, the ether wasremoved, and the residue was chromatographed with a gradient ofhexanes:ethyl acetate (100:0 to 20:1) to give 9 g (52%) of a clearliquid.

D. 3-Dodecyloxy-2-decyloxy-1-propanol

Detritylation of the product of Section C was accomplished usingp-toluenesulfonic acid (0.9 g) in CHCl₃:MeOH (72 mL:36 mL) (stirred atroom temperature for 48 hours, added 10 percent sodium bicarbonate,extracted with CHCl₃, dried over magnesium sulfate, and concentrated).The residue was purified by column chromatography using a gradient ofhexanes:ethyl acetate (20:1 to 5:1) to give 3.5 g (63%) of pure3-dodecyloxy-2-decyloxy-1-propanol.

E. 3-Dodecyloxy-2-decyloxypropyl Diphenyl Phosphate

Diphenylchlorophosphate (0.7 mL, 3.4 mmol) in 10 mL of diethyl ether wascooled to 4° C. under nitrogen. 3-Dodecyloxy-2-decyloxy-1-propanol (1.0g, 2.6 mmol) in 15 mL of pyridine and 5 mL of diethyl ether was added.The solution was warmed to room temperature then heated to about 52° C.for 3 hours. It was then cooled to room temperature, diluted with 50 mLof diethyl ether, and washed with water (2×25 mL), 0.5 N HCl (25 mL),and then water (25 mL). The organic layer was dried over sodium sulfate,filtered, and concentrated in vacuo to an oil. Chromatography with agradient of hexanes:ethyl acetate (10:1 to 1:1) produced 980 mg (1.5mmol, 60%) of pure product.

F. 3-Dodecyloxy-2-decyloxpropyl Phosphate

PtO₂ (69 mg) was placed in a Parr hydrogenation bottle. The diphenylphosphate of Section E (500 mg) in 100 mL of EtOH was then added. Thereaction mixture was hydrogenated at 15 psi for 1.5 hours until hydrogenuptake ceased. The reaction mixture was then filtered through Celite,and the EtOH was removed in vacuo. The oil was dissolved in 25 mL ofpyridine, concentrated in vacuo, and dried under high vacuum to give 350mg of pure solid phosphatidic acid.

G. 3′-Azido-3′-deoxy-5′-(3-dodecyloxy-2-decyloxypropyl)-phosphothymidine

AZT (43 mg, 0.16 mmol) and the phosphatidic acid of Section F (105 mg,0.22 mmol) were azeotropically dried with pyridine (3×3 mL) by in vacuoremoval. Dicyclohexylcarbodiimide (220 mg, 1.07 mmol) was added, and thedrying was repeated 4 times. A final 3 mL portion of pyridine was added,and the reaction mixture was stirred at room temperature in a desiccatorfor 4 days. Water (1 g) was added, and the mixture was stirred for 4hours. The solvents were removed in vacuo, and the crude material waschromatographed on 2 g of silica gel using a gradient of CHCl₃:MeOH(15:1 to 2:1). The product was dissolved in 11 mL of CHCl₃:MeOH:H₂O(4:6:1) and stirred with 1.5 g of Whatman preswollen microgranularcation (Na⁺) exchange concentrated in vacuo to give 37 mg of product(22%). FAB ms showed a [MH+Na] ion at 752.4350 (C₃₅H₆₄N₅O₉PNa, 1.4 ppm)and a [M+2Na]⁺ ion at 774.4179 (C₃₅H₆₃N₅O₉PNa₂, 2.0 ppm).

EXAMPLE 7 Procedure for Assessing Anti-HIV-1 Activity

The inhibitory effects of synthetic phospholipid compounds on thereplication of human immunodeficiency virus type 1 (HIV-1) virus incells was examined by the plaque assay procedure of L. Kucera et al.,Aids Research and Human Retroviruses 6, 491 (1990). In brief, CEM-SScell monolayers were infected with HIV-1. Infected cells were overlaidwith RPMI-1640 medium plus 10 percent fetal bovine serum (FBS)supplemented with different concentrations of inhibitor. Plaques werecounted at five days after infection. In this assay HIV-1 syncytialplaques are seen as large, multicellular foci (10 to 25nuclei/syncytium) that appear either brown and granular or clear. Sincethe number of HIV-1 syncytial plaques correlates with reversetranscriptase (RT) and p24 core antigen activity in the HIV-1 infectedcell overlay fluids, the syncytial plaque assay can be used to quantifythe amount of infectious virus. Reverse transcriptase activity wasassayed according to a described procedure (B. J. Poeisz et al., Proc.Natl. Acad. Scie. (U.S.A.) 77, 7415 (1980)). The activity of p24 coreantigen induced by HIV-1 infection of CEM-SS cells was measuredspectrophotometrically using the commercial Coulter EIA.

EXAMPLE 8 Results of Assessment of Anti-HIV-1 Activity

The results (Table 1) showed that all of the lipid compounds tested havean IC₅₀ against HIV-1 syncytial plaque formation ranging from 0.11 to0.64 μM. The compounds' IC₅₀ for cell cytotoxicity ranged from 11.85 to75.7 μM. The highest differential selectivity (611.7), which is a ratioof the cytotoxicity to the anti-HIV-1 activity, was obtained withcompound CP-130.

TABLE 1 Evaluation of Ether Lipids for Cytotoxicity and Anti-ViralActivity in CEM-SS Cells IC50 (μM) Differential Compounds CytotoxicityAnti-HIV-1 Activity Selectivity CP-128 31.6 0.14 225.7 CP-129 75.7 0.64176.0 CP-130 67.2 0.11 611.7 CP-131 36.6 0.32 114.2 JM-1 (cis) 11.850.42 28.2 Cytotoxicity was measured by uptake of TdR-H³ into total DNAin the presence of serial concentrations of compound. Anti-HIV-1activity was measured by standard plaque assay using CEM-SS cellmonolayers. Differential selectivity was determined by dividing the IC50for cytotoxicity by the IC50 for anti-HIV-1 activity.

EXAMPLE 9 Assessment of HBV Activity Inhibition

Human hepatoblastomas (HepG2) cells were tranfected with plasmid DNAcontaining tandem copies of HBV genomes. These cells constituitivelyreplicate HBV particles. HepG2 cells were treated with varyingconcentrations of CP-128 to determine the toxic cell concentration(TC₅₀) by neutral red dye uptake. Also, the inhibitory concentration(IC₅₀) of CP-128 for HBV replication was determined by ELISA.

It was determined that CP-128 cytotoxicity (TC₅₀) was 61.7 μM and theanti-HIV-1 activity (IC₅₀) was 15.6 μM (Table 1). These data indicatethat CP-128 has selective anti-HBV activity. Mechanism studies indicatethat CP-128 can have an inhibitory effect on the cellular production ofHBV-induced DNA, core antigen (HBcAg) and “e” antigen (HBeAg). As aresult, it is postulated that CP-128 and other compounds of the presentinvention are likely inhibiting the assembly of HBV nucleocapids and thepackaging of viral pregenomic DNA.

The foregoing examples are illustrative of the present invention and arenot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

1. A method of treating a viral infection in a subject in need of suchtreatment comprising administering to said subject an effectiveinfection-combating amount of a compound of3′-azido-3′-deoxy-5′-(3-dodecanamido-2-decyloxypropyl)-phosphothymidine,or a pharmaceutical salt thereof, wherein the viral infection comprisesa virus selected from the group consisting of HIV-1, herpes virus,influenza, respiratory syncytial virus, mumps, measles, andparainfluenze virus.
 2. The method according to claim 1, wherein saidviral infection is caused by HIV-1 virus.
 3. The method according toclaim 1, wherein said viral infection is caused by herpes simplex virus.4. The method according to claim 1, wherein said subject is a human. 5.The method according to claim 1, wherein said administration is oral. 6.The method according to claim 1, wherein said administration is rectal.7. The method according to claim 1, wherein said administration istopical.
 8. The method according to claim 1, wherein said administrationis intrathecal.
 9. The method according to claim 1, wherein saidadministration is buccal.
 10. The method according to claim 1, whereinsaid administration is parenteral.
 11. The method according to claim 1,wherein said compound is administered in an amount of at least about 0.1ng/kg to about 1 ng/kg.
 12. The method according to claim 1, whereinsaid compound is administered in an amount of at least about 0.001 μg/kgto about 0.1 g/kg.
 13. A method of treating a viral infection in asubject in need of such treatment comprising administering to saidsubject an effective infection-combating amount of a compound of3′-azido-3′-deoxy-5′-(3-dodecyloxy-2-decyloxypropyl)-phosphothymidine ora pharmaceutical salt thereof, wherein the viral infection comprises avirus selected from the group consisting of HIV-1, herpes virus,influenza, respiratory syncytial virus, mumps, measles, andparainfluenze virus.
 14. The method according to claim 13, wherein saidviral infection is caused by HIV-1 virus.
 15. The method according toclaim 13, wherein said viral infection is caused by herpes simplexvirus.
 16. The method according to claim 13, wherein said subject is ahuman.
 17. The method according to claim 13, wherein said administrationis oral.
 18. The method according to claim 13, wherein saidadministration is rectal.
 19. The method according to claim 13, whereinsaid administration is topical.
 20. The method according to claim 13,wherein said administration is intrathecal.
 21. The method according toclaim 13, wherein said administration is buccal.
 22. The methodaccording to claim 13, wherein said administration is parenteral. 23.The method according to claim 13, wherein said compound is administeredin an amount of at least about 0.1 ng/kg to about 1 ng/kg.
 24. Themethod according to claim 13, wherein said compound is administered inan amount of at least about 0.001 μg/kg to about 0.1 g/kg.